This invention relates to a reagent and a method for classification and counting of leukocytes by flow cytometry.
In the field of laboratory examination, the classification and counting of leukocytes can give very useful information for diagnosis of disease. For example, leukocytes in the normal peripheral blood usually include five normal types of leukocytes, i.e., lymphocytes, monocytes, neutrophils, eosinophils, and basophils, in constant proportions. In the presence of disease, the proportions of these leukocytes may vary. Measuring the proportions of the different types of leukocytes by classifying and counting these normal leukocytes is useful for obtaining information on the presence of disease.
Depending on the type of disease, immature leukocytes or erythrocytes which usually exist in the bone marrow and not in the peripheral blood, for example, immature granulocytes such as myeloblasts, promyelocytes, myelocytes or metamyelocytes, and erythroblasts, may appear in the peripheral blood in addition to these normal leukocytes. Abnormal leukocytes, such as lymphoblasts, atypical lymphocytes or abnormal lymphocytes, may also emerge. Detection of these cells, and their classification and counting, are of utmost importance for diagnosis of disease.
For leukocyte classification, it has been customary practice to prepare a blood smear, stain it appropriately, and observe the stained sample microscopically for classification and counting. In recent years, various full-automated differential leukocyte counters based on the principle of a flow cytometer have been provided. These devices permit highly precise classification of normal leukocytes, but did not make it possible to classify and count the above-mentioned abnormal cells, such as immature leukocytes, simultaneously with the classification and counting of normal leukocytes.
For instance, a reagent and a method which detect the appearance of immature leukocytes highly precisely by the principle of RF/DC measurement have been provided (Japanese Unexamined Patent Publication No. 6-273413). The method of this publication measures immature leukocytes electrically by utilizing their nature that under particular conditions, they are less destructible than normal leukocytes. It has also been suggested that their measurement can be made based on information on scattered light from them. However, this method aims only at the detection of immature leukocytes, and has excellent performance in their detection, but cannot classify and count normal leukocytes simultaneously with the measurement of immature leukocytes. Alternative methods should be performed for the classification and counting of normal leukocytes.
A different method that has been provided uses the principle of fluorometry to classify and count various immature leukocytes simultaneously with classifying leukocytes into four different populations (Japanese Unexamined Patent Publication No. 6-207942). With this method, normal leukocytes can be classified into only four populations at the same time, and basophils need to be measured separately in a different manner. A plurality of fluorescence signals also need to be measured, requiring a complicated, expensive apparatus.
A method for classifying and counting various immature leukocytes at the same time as the classification of leukocytes into five populations has also been provided (Japanese Unexamined Patent Publication No. 5-34251). This method requires a complicated detector for the classification of leukocytes into five populations. Two types of fluorescence information are also needed, thus involving a large, expensive apparatus.